CN1323199A - Liposome-entrapped topoisomerase inhibitors - Google Patents

Abstract

A composition for administration of a therapeutically effective dose of a topoisomerase inhibitor I or topoisomerase I/II inhibitor is described. The composition includes liposomes having an outer surface and an inner surface defining an aqueous liposome compartment, and being composed of a vesicle-forming lipid and of a vesicle-forming lipid derivatized with a hydrophilic polymer to form a coating of hydrophilic polymer chains on both the inner and outer surfaces of the liposomes. Entrapped in the liposomes is the topoisomerase inhibitor at a concentration of at least about 0.10 mumole drug per mumole lipid.

Description

Liposomal encapsulated topoisomerase enzyme inhibitor

Invention field

The present invention relates to contain the liposome composition of entrapped topoisomerase enzyme inhibitor.

Background of invention

In the U.S., cancer is the main dead factor that is only second to heart disease, and annual dead person surpasses 500,000.(Katzung, B., " Basic and Clinical Pharmacology ", the 7th edition, Appleton ﹠amp; Lange, Stamford CT, 1998, the 882 pages).In the present Therapeutic Method, 1/3rd patient adopts partial approach treatment as operation or radiotherapy, and this is effective really when tumor does not spread.Early diagnosis can make the patient's cure rate that adopts topical therapeutic improve.Yet in many cases, early stage little diffusion is a feature of tumor, and this shows needs for example chemotherapy of whole body therapeutic, with topical therapeutic and use for the control of tumor effective.

For between some diagnostic period with regard to whole or little diffusibility tumors of disseminating, chemotherapy is effective.These comprise carcinoma of testis, diffuse large cell lymphoma, Hodgkin's disease and choriocarcinoma and pediatric tumor such as acute lymphoblastic leukemia.For the other types of invasive cancer, chemotherapy provides a kind of alleviation but not curative therapy.Effectively the property alleviated treatment can temporarily be removed the symptom and the sign of cancer and be prolonged life.Nearest progress shows that chemotherapy is possible to the Chemical Control of certain cancers tumor.

A class medicine that is used for treatment of cancer is a topoisomerase enzyme inhibitor.These chemical compounds suppress the enzymatic activity of topoisomerases, and this enzyme participates in the duplicating of DNA, reparation, genetic recombination and transcribes.An example of topoisomerase enzyme inhibitor is a camptothecine, is the active native compound of a kind of interference type, and this enzyme relates to dna replication dna and rna transcription.Camptothecine and camptothecin analogues topotecan (topotecan) and irinotecan (irinotecan) have been approved for clinical.

Fracture by disturbing type/reconnect activity, camptothecine and analog thereof are effective to the chemotherapy of cancer.This chemical compound is stable and form a kind of reversible enzyme-camptothecine-DNA ternary complex, reconnects step during this complex can stop the topoisomerase enzyme reaction to interrupt splitting/connects to circulate.

The problem that camptothecine exists is a water-insoluble, and this has hindered transporting of medicine.Many camptothecin analogues have been prepared to improve the water-soluble of this compounds.Another problem of camptothecine and analog thereof is that its Alpha-hydroxy lactonic ring is easy to hydrolysis in water environment.The lactonic ring open loop forms the carboxylate form of medicine and shows as extremely low anti-type activity.

Existing many methods have been described the stability of improving camptothecine and analog thereof.One of them method is that chemical compound is encapsulated in the liposome.

Burke (US5552156) has described a kind of liposome composition, attempts to solve the unstability of camptothecine and analog thereof, and this method is that chemical compound is encapsulated in the liposome, and this liposome has a bilayer lipid membrane that allows the chemical compound infiltration, embeds.In chemical compound embedding duplicature, thereby it has avoided hydrolysis at the liposome center away from water environment.

A problem of this method is that liposome disposed fast by reticuloendothelial system (RES) from blood, so just stoped the gathering at tumor locus of transporting especially of medicine.

(Oncology Reseach such as Subramanian and Muller, 7 (9): 461-469 (1995)) described the Liposomal formulation of topotecan, but and reported with liposomal encapsulated form stable topology and replace health to avoid the inactivation that causes because of its lactonic ring hydrolysis.Yet the external biological activity of this liposomal encapsulated medicine only is 60% of a free drug.

Lundberg (Anti-Cancer Drug Design, 13:453 (1998)) has described two kinds of lipotropys, the oleate derivant of camptothecin analogues, and described camptothecin analogues is encapsulated in the liposome and embeds in the duplicature to stablize its lactonic ring.Daoud (Anti-Cancer Drug, 6:83-93 (1995)) has described and has comprised the liposome composition that embeds the camptothecine on the duplicature.These with reference in liposome be conventional method and make, promptly medicine is passive is encapsulated in the liposome and is stablized in bilayer lipid membrane medicine is hidden.The liposome that this method makes is difficult to the medicine of load q.s to reach clinical efficacy.

Therefore, this area still needs to have the Liposomal formulation of following characteristic: (ⅰ) comprise a kind of topoisomerase enzyme inhibitor, for example camptothecine and analog thereof; (ⅱ) in blood flow, can keep the long period; (ⅲ) keep active anticancer; (ⅳ) comprise the q.s clinical application thing of institute's load.

The invention summary

Correspondingly, an object of the present invention is to provide the topoisomerase enzyme inhibitor compositions that is used to improve treatment of cancer.

Another object of the present invention provides the liposome composition of the topoisomerase enzyme inhibitor administration that is used for antineoplaston.

On the other hand, the present invention includes a kind of compositions that is used for the treatment of patient tumors, it comprises the liposome of the vesicle-forming lipids of using the hydrophilic polymer derivatization that contains vesicle-forming lipids and 1-20 mole %.Described liposome forms under polymer can be distributed in condition on the liposome duplicature both sides.Seal in the liposome be type inhibitor or type/II inhibitor, its concentration is at least 0.10 micromole medicine/micromole's lipoid.Liposome has certain inside/outside ion gradient, and this gradient is enough to make the type inhibitor of specific concentrations or type/II inhibitor to be retained in the liposome.

In one embodiment, topoisomerase enzyme inhibitor is to be selected from the type inhibitor that comprises camptothecine and derivant thereof.For example, camptothecin derivative can be 9-aminocamptothecin, 7-ethyl-camptothecin, 10-hydroxycamptothecine, 9-nitrocamptothecin, 10,11-methylene-dioxy camptothecine, 9-amino-10,11-methylene-dioxy camptothecine or 9-chloro-10,11-methylene-dioxy camptothecine.

In another embodiment, camptothecin derivative is irinotecan, topotecan, (7-(4-methyl piperazine methylene)-10,11-ethylenedioxy-20 (S)-camptothecine, 7-(4-methyl piperazine methylene)-10,11-methylene-dioxy-20 (S)-camptothecine or 7-(2-N-isopropylamino) ethyl)-(20S)-camptothecine.

In another embodiment, topoisomerase enzyme inhibitor is type/II inhibitor, 6-[[2-(dimethylamino)-ethyl for example] amino]-3-hydroxyl-7H-indeno [2,1-c] quinoline-7-ketone dihydrochloride, azotoxin (azotoxin) or 3-methoxyl group-11H-pyridine [3 ', 4 '-4,5] pyrroles [3,2-c] quinoline-1, the 4-diketone.

Contained hydrophilic polymer can be polyvinylpyrrolidone, polyvinyl methyl ether, Ju Jia Ji oxazolidine, Ju Yi Ji oxazolidine, poly-Qiang Bing Ji oxazolidine, poly-hydroxypropyl methyl acrylamide, PMAm, polydimethylacrylamiin, polymethylacrylic acid hydroxypropyl acrylate, Poly(Hydroxyethyl Methacrylate), hydroxy methocel, hydroxyethyl-cellulose, Polyethylene Glycol and poly-asparagine in the liposome composition.

In preferred embodiments, hydrophilic polymer is that molecular weight is 500-5,000 daltonian Polyethylene Glycol.

In another embodiment, liposome comprises that further phase transition temperature is higher than 37 ℃ vesicle-forming lipids.

In another embodiment, vesicle-forming lipids is hydrogenated soy phosphatidyl choline, distearoylphosphatidylcholine or sphingomyelins.Preferred liposome composition contains the distearoylphosphatidylcholine of the poly-diethanol derivatization of usefulness of hydrogenated soy phosphatidyl choline, 1-20 mole % of 20-94 mole % and the cholesterol of 5-60 mole %.

Comprise the distearoylphosphatidylcholine of the poly-diethanol derivatization of usefulness of hydrogenated soy phosphatidyl choline, 5-20 mole % of 30-65 mole % and the cholesterol of 30-50 mole % in another preferred composition.

On the other hand, the present invention includes the compositions that is used for type inhibitor or type/II inhibitor administration, comprise the liposome that contains vesicle-forming lipids and have certain inside/outside ion gradient, this gradient is enough to make medicine to be retained in the liposome.What seal in the liposome is type inhibitor or type/II inhibitor, and its concentration is at least 0.20 micromole medicine/micromole's lipoid.

On the other hand, the present invention includes the method for treatment patient tumors, comprise that preparation contains the liposome of vesicle-forming lipids of the 1-20 mole % of useful hydrophilic polymer chains derivatization, described liposome forms under polymer can be distributed in condition on the liposome duplicature both sides.What seal in the liposome is type inhibitor or type/II inhibitor, and its concentration is at least 0.10 micromole/micromole's lipoid.Liposome has certain inside/outside ion gradient, and this gradient is enough to make the type inhibitor of specific concentrations or type/II inhibitor to be retained in the liposome.Give the experimenter with liposome then.

In the embodiment in this respect, method further comprise adopt remote control medicine carrying method (remoteloading) for example ammonium sulphate gradient type inhibitor or type/II inhibitor packages are enclosed in the liposome.

Read following detailed in conjunction with the accompanying drawings, these and other purpose of the present invention and feature will obtain more comprehensively understanding.

The accompanying drawing summary

The blood circulation time curve that Figure 1A compares with free drug (solid squares) for liposomal encapsulated MPE-camptothecine (filled circles).Percent with injected dose is the function of time.

Figure 1B be give the liposomal encapsulated MPE-camptothecine (filled circles) of rat and free (non-liposome) MPE-camptothecine (solid squares) back MPE-camptothecine blood drug level in time (hour) change curve.

Fig. 2 A for inoculation HT-29 colon tumor after mice body weight (g) in time (my god) change curve.Behind inoculated tumour the 10th, 16 and 23 day, give the MPE-camptothecine of the liposomal encapsulated MPE-camptothecine of animal 24mg/kg (filled circles), 15mg/kg (solid triangle) and 6mg/kg (solid squares) and 24mg/kg (open circles), 15mg/kg (hollow triangle) and 6mg/kg (square hollow) free form.

Fig. 2 B is gross tumor volume (mm behind the inoculation HT-29 colon tumor ³) in time (my god) change curve.Behind inoculated tumour the 10th, 16 and 23 day, give the MPE-camptothecine of the liposomal encapsulated MPE-camptothecine of animal 24mg/kg (filled circles), 15mg/kg (solid triangle) and 6mg/kg (solid squares) and 24mg/kg (open circles), 15mg/kg (hollow triangle) and 6mg/kg (square hollow) free form.

Fig. 3 A for inoculation HT-29 colon tumor after mice body weight (g) in time (my god) change curve.Behind inoculated tumour the 9th, 16 and 23 day, give the liposomal encapsulated MPE-camptothecine of animal 5mg/kg (hollow triangle), 3mg/kg (hollow inverted triangle), 1mg/kg (open diamonds), 0.5mg/kg (open circles) and 0.1mg/kg (square hollow) and the MPE-camptothecine of 20mg/kg free form.

Fig. 3 B is gross tumor volume (mm behind the inoculation HT-29 colon tumor ³) in time (my god) change curve.Behind inoculated tumour the 9th, 16 and 23 day, give the liposomal encapsulated MPE-camptothecine of animal 5mg/kg (hollow triangle), 3mg/kg (hollow inverted triangle), 1mg/kg (open diamonds), 0.5mg/kg (open circles) and 0.1mg/kg (square hollow) and the MPE-camptothecine of 20mg/kg free form.

Fig. 4 A-4B be give the liposomal encapsulated topotecan of rat (solid triangle) and free (non-liposome) topotecan (solid squares) back topotecan plasma concentration versus time (hour) change curve.Fig. 4 A and Fig. 4 B give 2mg/kg and 5mg/kg respectively.

Fig. 5 A for inoculation HT-29 colon tumor after mice body weight (g) in time (my god) change curve.Behind inoculated tumour the 9th, 16 and 23 day, give animal 2mg/kg (rhombus), 5mg/kg (circle) and the liposomal encapsulated topotecan of 8mg/kg (square hollow); The MPE-camptothecine (triangle) that 4mg/kg is liposomal encapsulated; Topotecan of 25mg/kg free form (inverted triangle) and saline (solid squares).

Fig. 5 B is gross tumor volume (mm behind the inoculation HT-29 colon tumor ³) in time (my god) change curve.In inoculating back the 9th, 16 and 23 day, give animal 2mg/kg (rhombus), 5mg/kg (circle) and the liposomal encapsulated topotecan of 8mg/kg (square hollow); The MPE-camptothecine (triangle) that 4mg/kg is liposomal encapsulated; Topotecan of 25mg/kg free form (inverted triangle) and saline (solid squares).

Fig. 6 be give the liposomal encapsulated CKD602 of rat (filled circles) and free (non-liposome) topotecan (solid squares) back CKD602 plasma concentration versus time (hour) change curve.

Fig. 7 A for inoculation HT-29 colon tumor after mice body weight (g) in time (my god) change curve.Behind inoculated tumour the 9th, 16 and 23 day, give animal 4mg/kg (rhombus), 2mg/kg (circle) and the liposomal encapsulated CKD602 of 1mg/kg (square hollow); The MPE-camptothecine (triangle) that 4mg/kg is liposomal encapsulated; CKD602 of 20mg/kg free form (inverted triangle) and saline (solid squares).

Fig. 7 B is gross tumor volume (mm behind the inoculation HT-29 colon tumor ³) in time (my god) change curve.In inoculating back the 9th, 16 and 23 day, give animal 4mg/kg (rhombus), 2mg/kg (circle) and the liposomal encapsulated CKD602 of 1mg/kg (square hollow); The MPE-camptothecine (triangle) that 4mg/kg is liposomal encapsulated; CKD602 of 20mg/kg free form (inverted triangle) and saline (solid squares).

Detailed description 1. definition of invention

Unless refer else, following term has following implication.

" effective dose " or " effective dose " refers to suppress the undesirable cell growth and for example stops the undesirable cell growth or reduce existing Growth of Cells such as the required amount of growth of tumour cell. This effective dose is subjected to factor affecting known in the art, such as the type of Growth of Cells, the methods of administration, experimenter's age, the seriousness of Growth of Cells etc. Those skilled in the art can take into account these factors and determine corresponding effective dose.

" effective antitumour therapy in the treatment " refers to effectively to keep or to reduce the size of primary tumo(u)r or metastatic tumour such as the methods for the treatment of of volume.

" topoisomerase I inhibitor " refers to suppress or to reduce any compound of the activity of topoisomerase I.

" topoisomerase I/II inhibitor " refers to suppress simultaneously or to reduce any compound of the activity of topoisomerase I and type Ⅱ topoisomerase.

" topoisomerase enzyme inhibitor " refers to topoisomerase I inhibitor or topoisomerase I/II inhibitor.

" MPE-camptothecine " refers to 7-(4-methyl piperazine methylene)-10,11-ethylenedioxy-20 (S)-camptothecine.

" TPT " refers to 9-dimethyl-aminomethyl-10-hydroxycamptothecine.

" CKD-602 " refers to 7-(2-N-isopropylamino) ethyl)-(20S)-camptothecine. II. liposome composition

The present invention relates to the liposome composition for topoisomerase I inhibitor or topoisomerase I/II inhibitor administration. In supporting experiment of the present invention, be encapsulated in following three kinds of topoisomerase enzyme inhibitors in the liposome and studied its body internal characteristic: TPT, 7-(4-methyl piperazine methylene)-10,11-ethylenedioxy-20 (S)-camptothecine (referring to " MPE-camptothecine " herein) and 7-(2-N-isopropylamino) ethyl)-(20S)-camptothecine (referring to " CKD-602 " herein). As will be described below, adopt remote control medicine carrying method with drug encapsulation in liposome so that higher drug loading stably be retained in the liposome. Experimental results show that in the body of preparation: compare with the topoisomerase enzyme treatment with free form, liposome composition has improved therapeutic activity astoundingly. More specifically, as will be described below, the dosage that reaches the required liposomal encapsulated topoisomerase I inhibitor MPE-camptothecine of therapeutic anti-cancer therapies is wanted low 20 times of specific ionization drug dose.

In this section, will describe liposome composition, comprise the preparation method of liposome.

A. the composition of liposome

Be applicable to the liposome that liposome of the present invention comprises that those mainly are comprised of vesicle-forming lipids. Vesicle-forming lipids for example phosphatide can spontaneous formation bilayer vesicles in water. Liposome also can comprise the some other lipoid that mixes double-layer of lipoid, and these lipoids have hydrophobic group, the duplicature hydrophobic region that links to each other with inside and the headgroup that stretches to outside, duplicature polar surfaces.

Vesicle-forming lipids preferably has two hydrocarbon chains, typically is acyl chain, and a polarity or nonpolar headgroup. Many synthetic and natural vesicle-forming lipids are arranged, comprise phosphatide, such as lecithin, phosphatidyl-ethanolamine, phosphatidic acid, the pure and mild sphingomyelin of phosphatidyl-4, wherein typically two hydrocarbon chains have 14-22 carbon atom and have different saturation degrees. Lipoid and phosphatide with different saturation acyl chain described above can be buied or be made by disclosed method by commercially available. Other suitable lipoids comprise glycolipid and sterol such as cholesterol.

Cation lipoid also is applicable to liposome of the present invention as the less important main or unique composition in the lipid composition. This type of cation lipoid typically has a lipophilic portion such as sterol, acyl group or diacyl chain, wherein the clean positive electricity of lipoid lotus. Preferably, the headgroup lotus positive electricity of lipoid. Exemplary cation lipoid comprises 1,2-, two oil base oxygen base-3-(trimethyl is amino) propane (DOTAP); N-1-(2,3 ,-two myristyl oxygen) propyl group]-N, N-dimethyl-N-ethoxy ammonium bromide (DMRIE); N-1-(2,3 ,-two oil base oxygen) propyl group]-N, N-dimethyl-N-ethoxy ammonium bromide (DORIE); N-[1-(2,3-, two oil base oxygen) propyl group]-N, N, N-trimethyl ammonium chloride (DOTMA); 3[N-(N ＇, N ＇-dimethyl aminoethyl) carbamoyl] cholesterol (DC-Chol); And dimethyl dioctadecyl ammonium (DDAB).

But the cation lipoid of encystation also can be neutral lipoid, and for example DOPE (DOPE) or amphipathic lipoid such as phospholipid are used cation lipoid such as many lysins or other polyamine lipoid derivatization.For example neutral lipoid (DOPE) is that available many lysins are derived and are cation lipoid.

In another embodiment, select flowability or the rigidity of vesicle-forming lipids to reach specific degrees, purpose is the rate of release of the stability of control liposome in serum and control liposome institute entrapped drug.

By mixing the lipoid of relative stiffness, higher phase transition temperature is for example arranged as being higher than room temperature, preferably being higher than body temperature and until the lipoid of 80C, can make liposome have more inflexible double-layer of lipoid or iquid crystalline bilayer.Inflexible for example saturated lipoid makes bilayer lipid membrane have more rigidity.Known other lipoid compositions for example cholesterol also have contribution to the membrane rigidity of double-layer of lipoid structure.

On the other hand, by mixing relatively mobile lipoid, typically has lower liquid state-liquid crystal state phase transition temperature is improved lipoid as the lipoid that is equal to or less than room temperature, more preferably is equal to or less than the lipoid phase of body temperature flowability.

Main phase transition temperature is about the liposome basis that 2-80 ℃ vesicle-forming lipids is suitable for use as the present composition.In a preferred embodiment, adopt main phase transition temperature to be higher than 37 ℃ vesicle-forming lipids as the basic lipoid composition of liposome.In another preferred embodiment, adopted phase transition temperature 37-70 ℃ lipoid.As example, the main phase transition temperature of lipoid distearoylphosphatidylcholine (DSPC) is 55.1 ℃, and the phase transition temperature of lipoid hydrogenated soy phosphatidyl choline (HSPC) is 58 ℃.The phase transition temperature data of many lipoids are all tabulated in multiple publication, for example in Avanti polarity lipoid catalogue and lipoid heat to phase change data (Lipid, Nist standard reference data 34).

Liposome also comprises the vesicle-forming lipids with the hydrophilic polymer derivatization.Described at US5013556 and WO98/07409, be hereby incorporated by.This hydrophilic polymer provides a kind of hydrophilic polymer chains surface coatings for the bilayer lipid membrane surfaces externally and internally of liposome.Outermost hydrophilic polymer chains coating makes liposome have longer blood circulation time in vivo.The aqueous compartment that the hydrophilic polymer chains coating of internal layer then stretches to liposome is between the double-layer of lipoid and the central core chamber interior, and contacts with the institute encapsulation compound after by remote control medicine carrying load chemical compound.Will illustrate as following, the hydrophilic polymer chains coating is distributed in the inside and outside Liposomal formulation of surface of liposome, will be used for providing type inhibitor that chemical compound is retained in liposome or type/II inhibitor combination to improve therapeutic activity.

Be suitable for comprising any above-mentioned listed lipoid, be specially phospholipid such as dioleoyl PHOSPHATIDYL ETHANOLAMINE (DSPE) with the vesicle-forming lipids of hydrophilic polymer derivatization.

Be suitable for the hydrophilic polymer of vesicle-forming lipids derivatization is comprised polyvinylpyrrolidone, polyvinyl methyl ether, Ju Jia Ji oxazolidine, Ju Yi Ji oxazolidine, poly-Qiang Bing Ji oxazolidine, poly-hydroxypropyl methyl acrylamide, PMAm, polydimethylacrylamiin, polymethylacrylic acid hydroxypropyl acrylate, Poly(Hydroxyethyl Methacrylate), hydroxy methocel, hydroxyethyl-cellulose, Polyethylene Glycol and poly-asparagine.Above-mentioned polymer can be used as homopolymer or block or random polymer.

A kind of preferred hydrophilic chain is Polyethylene Glycol (PEG), and preferred PEG chain molecular weight is 500-10,000 dalton, more preferably 500-5,000 dalton, most preferably 1,000-2,000 dalton.Also preferably can commercially availablely buy have the multiple polymers size with the end capped PEG analog of methoxy or ethoxy as hydrophilic polymer, 120-20 for example, 000 dalton.

The existing description of relevant preparation method, for example US5395619 with hydrophilic polymer derivatization vesicle-forming lipids.Preparation contains also existing description of method of the liposome of this kind derivatization lipoid, wherein typically comprises the rub derivatization lipoid of ear % of 1-20 in the Liposomal formulation.Understand easily, hydrophilic polymer can stably be incorporated on the lipoid with the lipoid coupling or with a kind of unsettled bond, and this key makes and by the liposome of coating the polymer chain coating split away off from liposome.

B. topoisomerase enzyme inhibitor

Liposome of the present invention comprises a kind of topoisomerase enzyme inhibitor that is encapsulated in the liposome.Seal is to attempt drug encapsulation in the aqueous core and aqueous space of liposome.Understand easily, have certain hydrophobic chemical compound and also can be encapsulated in the double-layer of lipoid of liposome.

The supercoiled introducing of topoisomerase catalytic dna (introduction) and lax.Known several types have not homospecific enzyme for the duplicating of DNA, reparation, gene recombinaton with transcribe very important.The simplest topoisomerase of the type by name supercoiled DNA that can relax, this is a process spontaneous aspect energy.But the introducing that the gyrase catalytic dna negative supercoiling that is known as type reverses, this process need energy and be that ATP relies on.In the dna replication dna process, type and II all have lax by the function of unwindase at the positive supercoiling of replication fork the place ahead introducing.In addition, gyrase can be introduced negative twist at the fragment place that single stranded DNA occurs changes.

Topoisomerase enzyme inhibitor is to suppress the active chemical compound of topoisomerase.The known active chemical compound of type that suppresses is used as the type inhibitor, is used as topoisomerase II inhibitor and can suppress the active chemical compound of type.Some are known to have the type of inhibition and the active chemical compound of type is used as type/II inhibitor simultaneously.

Being preferred for type inhibitor of the present invention is camptothecine and analog thereof.Camptothecine is five-membered ring alkaloid (Wall, M, the E. etc. that separate from the wood of Chinese plant camptotheca acuminata and bark at first.J.Am.Chem.Soc.,94:388(1996))。Camptothecine is owing to irreversibly suppressing the active pharmacotoxicological effect that shows of type.The synthetic method of camptothecine and analog thereof or derivant has belonged to known, and is summed up and enumerate in US5244903, is hereby incorporated by.

The analog of camptothecine comprises SN-38, and ((+)-(4S)-4,11-diethyl-4,9-dihydroxy-1H-pyrans are [3 ＇, 4 ＇: 6,7]-indolizino [1,2-b] quinoline-3,14 (4H, 12H)-diketone) also; 9-aminocamptothecin; Topotecan (hycamtin, 9-dimethyl-aminomethyl-1,2 0-hydroxy camptothecin); Irinotecan (CPT-11,7-ethyl-10-[4-(1-piperidines)-1-piperidines]-ketonic oxygen base-camptothecine), be hydrolyzed to SN-38 in vivo); 7-ethyl-camptothecin and derivant thereof (Sawada, Chem.Pham.Bull such as S., 41 (2): 310-313 (1993)); 7-chloromethyl-10,11-methylene-dioxy base-camptothecine; And other (SN-22, Kunimoto, T. etc.J.Pharmacobiodyn., 10 (3): 148-151 (1987); N-formamido group-12,13, dihydro-1 ,-11 dihydroxy-13-(β-D-glucopyranose)-H-indole [2,3-a] pyrrolo-[3,4-c] carbazoles-5,7 (6H)-diketone (NB-506, Kanzawa, F. etc.Cancer Res., 55 (13): 2806-2813 (1995); DX-8951f and lurtotecan (GG-211 or 7-(4-methyl piperazine-methylene)-10,11-ethylenedioxy-20 (S)-camptothecine) (Rothenberg, M.L., etc.Ann.Oncol .8 (9): 837-855 (1997)) and 7-(2-N-isopropyl amino) ethyl)-(20S)-camptothecine (CKD602, Chong Kun Dang company, Korea S).

Have the type of inhibition and the active topoisomerase enzyme inhibitor of type simultaneously and comprise 6-[[2-(dimethylamino)-ethyl] amino]-3-hydroxyl-7H-indeno [2,1-c] quinoline-7-ketone dihydrochloride, (TAS-103, Utsugi, t., etc.Jpn.J.Cancer Res., 88 (10) 992-1002)) and 3-methoxyl group-11H-pyrido [3 ＇, 4 ＇-4,5] pyrroles [3,2-c] quinoline-1, the 4-diketone (AzalQD, Riou, J.F., etc.Mol.Pharmacol.,40(5):699-706(1991))。

In one embodiment of the invention, the type inhibitor that is used for administration is the pharmacologically active enantiomer with camptothecin analogues of a chiral centre.Can use technology well known by persons skilled in the art from racemic mixture, to split enantiomer.

C. the preparation method of liposome composition

Liposome can be by the preparation of many technology, as Szoka, and F., Jr. waits and describes among Ann.Rev.Biophys.Bioeng.9:467 (1980), will describe the specific embodiment of support liposome preparation of the present invention below.Typically, liposome is the multicell folliculus (MLVs) with the preparation of simple lipid film hydration technology.In this method, form the lipoid of liposome and comprise that a kind of mixture of vesicle-forming lipids of hydrophilic polymer derivatization is dissolved in the The suitable solvent, forms dry film behind the evaporating solvent in container.Add aqueous medium then film is covered formation MLVs, typically size is the 0.1-10 micron.The exemplary method that is used to prepare the derivatization lipoid and form the polymer coating liposome is seen the US5013556,5631018 and 5395619 that owns together, is hereby incorporated by.

Select can be incorporated into medicine on the liposome by standard method, comprise (ⅰ) passive water soluble compound of sealing of aqueous solution hydrated lipidic film with medicine, (ⅱ) hydration contains the passive lipophilic compound of sealing of lipid film of medicine, (ⅲ) by the medicine of liposome inside/outside ion gradient load tool ionization character, term is " a remote control medicine carrying method ".Other preparation methoies such as reverse evaporation phase liposome method also are suitable for.

The present invention preferably adopts remote control medicine carrying legal system to be equipped with liposome.In the research of carrying out for support the present invention, adopt remote control medicine carrying method that three kinds of exemplary type inhibitor are loaded in the preformed liposome by ion concentration gradient, as describing among the prior art US 5192549 and describing as embodiment 1.Compare with other carrying methods, it is high that the drug level that the liposome central compartment that remote control medicine carrying method makes gathers is wanted.In a preferred embodiment of the invention, at least about 0.10 micromole medicine/micromole's lipoid, more preferably at least about 0.15 micromole medicine/micromole's lipoid, most preferably type inhibitor or the type/II inhibitor at least about 0.20 micromole medicine/micromole's lipoid loads in the liposome.Support liposome of the present invention to comprise MPE-camptothecine, topotecan or CKD602.Will discuss as embodiment 1, load to these compound concentrations in the liposome greater than 0.20 micromole medicine/micromole's lipoid (referring to the table of embodiment 1) by remote control medicine carrying method.

Be used for having of remote control medicine carrying stride the liposome of the ion gradient of liposome bilayer can be by the preparation of many technology.Described a typical method above, the lipid mixture that wherein can form liposome is dissolved in the suitable organic solvent and evaporation formation thin film in container.Cover the thin film that forms with the aqueous medium that contains form the solute of water in the liposome interior space then.

After liposome forms, adopt known technology vesicle granulating (sizing) (pressing the certain size manufacturing) to be obtained the liposome of desired particle size range.It is the 0.04-0.25 micron that preferred liposome is made particle diameter equably.Adopt ultrasonic or homogenize is handled liposome and can be made the small unilamellar vesicle that typical particle diameter is the 0.04-0.08 micron (SUVs).Adopt the film squeezing and pressing method to be about to liposome, can make the even liposome of particle diameter for about 0.08-0.4 micron by 0.03-0.5 micron, typically 0.05,0.08,0.1 or 0.2 micron polycarbonate membrane or other films that definite aperture is arranged.The aperture of film with approximately conformed to by pushing the liposome maximum particle diameter that film generates, particularly when prepared product by twice or repeatedly pushed under the situation of same film all the more so.Granulating preferably carries out at the original buffer that is used for the lipid hydration, and the inner phase of liposome remains with this medium all the time in preparation process like this.

Behind the granulating, handle the external agency of liposome and stride the liposome membrane ion gradient with generation, low in typically being/outer high Concentraton gradient.Several different methods can reach this purpose, for example (ⅰ) dilutes external agency, (ⅱ) to required final medium dialysis, (ⅲ) with required medium by molecular sieve chromatography such as Sephadex G-50, or (ⅳ) more sedimentary liposome is suspended in the required final medium behind the high speed centrifugation.The selection of recognizing external agency is at present got and is picked out the mechanism that forms in gradient and required outside pH value.

In producing the straightforward procedure of ion gradient, hydration, granulated liposome have the interior media pH value of a selection.The titration liposome turbid liquor, or apparatus has the buffer of required outside pH value to exchange foreign minister's buffer as stated above until reaching required final pH value.For example, the pH value that resembles original medium in the so selected buffer of glutamic acid or phosphate buffer is 5.5, and final external agency is the identical or different buffer of pH value 8.5.Preferably contain the inside and outside medium of roughly the same osmolarity, for example buffer, salt or low molecular weight solutes such as sucrose concentration are suitably regulated.

In another method, produce gradient by in liposome, comprising a kind of selected ionophore.In order to illustrate, preparation contains the liposome of valinomycins in the liposome bilayer in the potassium buffer, and granulating is used the sodium buffer-exchanged then, produces the gradient of an inner potassium/outside sodium.Potassium ion mobile phase from inside to outside should produced inside low/gradient of outside high pH, infer this be the proton that produces corresponding to net negative charge passes lipid film to liposome move (Deamer, etc.1972)。

In another preferred method, be to be used for the proton gradient of drug loading with generation, described in US5192549 by setting up an ammonium ion gradient of striding liposome membrane.The liposome here prepares in containing the aqueous buffer solution of ammonium salt, typically is ammonium salt such as the ammonium sulfate of 0.1-0.3M, suitable pH value such as 5.5-7.5.Available Sulfated polymer produces gradient, as dextran ammonium sulfate or heparin sulfate.Behind liposome formation and the granulating, with the Medium Exchange external agency that does not contain ammonium ion, for example same buffer, but ammonium sulfate wherein can be made liposome keep the sodium chloride or sugared replacement of identical inside and outside osmolarity.

After liposome formed, the ammonium ion of liposome interior and ammonia and proton reached balance.Ammonia can permeate double-layer of lipoid and overflow from liposome interior.The continuous effusion of ammonia makes the intravital balance of lipid move to the positive balance that produces proton.

By in the liposome turbid liquor that medicine is joined the tool ion gradient topoisomerase enzyme inhibitor being loaded in the liposome, handle suspension guaranteeing that chemical compound can be entered by external agency under the condition in the liposome effectively.The incubation conditions following (ⅰ) that is fit to drug loading allows derived compounds to diffuse to liposome with a kind of uncharged form, (ⅱ) preferably reach higher drug loading concentration, as entrapped drug 5-500mM, more preferably 20-200mM, 50-300mM most preferably.

Preferably under the temperature that is higher than liposome lipoid phase transition temperature, carry out load.Therefore, for the liposome that is mainly formed by saturated phospholipid, load temperature can be at 60 ℃ or higher.Typically load time is 15-200 minute, and this depends on the relative concentration of medicine to the double-deck permeability of the membrane of liposome, temperature and liposome lipoid and medicine.

By to liposome concentration, add the outside concentration of chemical compound and the suitable selection of ion gradient, but all basically chemical compounds all load go into liposome.For example, the gradient of 3 pH units (or such gradient intensity of employing ammonium ion gradient) can make the final inside and outside concentration ratio of medicine be about 1000: 1.The Cmax of the internal lipids body volume of known calculations and institute's carrying medicament just can be selected the amount of external agency Chinese medicine, and medicine can be loaded in the liposome substantially fully like this.

In addition, if drug loading can not consume the external agency of free drug effectively, can be at drug loading post processing liposome turbid liquor until removing non-encapsulated medicine.Free drug also can be by removing as molecular sieve chromatography, dialysis or centrifugal method.

In other embodiments, topoisomerase enzyme inhibitor loads in the preformed liposome, and this liposome interior contains and can effectively mix and improve the trapping agent (trapping agent) that chemical compound keeps in liposome with topoisomerase enzyme inhibitor.In preferred embodiments, trapping agent is a kind of multi-anion copolymer, for example preferably contains the molecule of the repetitive of similar chemical constitution and ionizing group, promptly ionizable generation ionic charge, is preferably the chemical functional group of negative charge.Molecular weight 400-2,000,000 daltonian polymer are all suitable.

Multi-anion copolymer is encapsulated in the liposome in the lipid vesicle forming process.In with the process of drug loading in the preformed liposome, polymer is used for medicine is held back or is retained in the liposome.In the research described here, dextran sulfate is as typical multi-anion copolymer.Dextran sulfate is the anhydroglucose polymer of about 2.3 sulfate groups/glucose residues.It contains α-D-(1-6) key of about 95% and makes the ramose residue of dextran (1-3) key.Obtaining molecular weight easily is 5,000-500,000 daltonian polymer.Yet other suitable polymers also comprise the hydrophilic polymer of sulphation, sulfonated, carboxylated or phosphorylation.For example, sulphuric acid proteoglycan such as heparin sulfate, sulfated polysaccharide such as sulfate cellulose or cellulose derivative, carrageenin, mucin, the sulphation polypeptide is as many lysins of being with sulphation amino, glycopeptide and the hyaluronic acid that has sulfonic acid derivitised carbohydrate or peptide subunit.Also comprise chondroitin sulfate A, B and C, keratin sulfate, dermatan sulfate.Polymer also can be to be modified into the neutral polymer that comprises the anionic functional group.For example, amylose, pectin, amylopectin, cellulose and be modified into the dextran that comprises the anion subunit.The polymer that has sulfo group also is suitable as polyvinyl sulfuric acid, polyvinyl sulfonic acid, polystyrolsulfon acid and sulphuric acid rosin gum.

Preparation contains liposome description to some extent in embodiment 4 of trapping agent.In this embodiment, be added in the dextran ammonium sulfate solution after the liposome lipoid being dissolved in ethanol, mix forming the liposome that is encapsulated with the dextran ammonium sulfate, multi-anion copolymer sulphuric acid dextran just is encapsulated in the liposome like this.Stride liposome ammonium ion gradient by what the exchange external agency was formed for the remote control medicine carrying.III. the compositions vivo medicine-feeding

Liposome of the present invention is supported in preparation shown in embodiment 1.Under the ammonium sulfate ion concentration gradient, with type inhibitor (7-(4-methyl piperazine)-methylene)-10,11-ethylenedioxy-20 (S)-camptothecine), refer to " MPE-camptothecine " at this; Topotecan; And 7-(2-(N-isopropyl amino) ethyl)-(20S)-camptothecine, refer to that at this " CKD-602 " loads in the liposome.Liposome is that the DSPE (PEG-DSPE) of 55.4: 39: 5.6 hydrogenated soy phosphatidyl choline, cholesterol and polyglycol derivatization is formed by mol ratio.Table among the embodiment 1 has been summed up the medicine of preparation Liposomal formulation and the ratio of lipoid.According to the volume of catching of extruding liposome is 0.9 microlitre/micromole's lipoid, and calculate three kinds of compound concentrations in the liposome: the MPE-camptothecine is 284mM, and topotecan is that 264mM and CKD-602 are 298mM.According to the volume of catching of extruding liposome is 1.5 microlitres/micromole's lipoid, and calculate the concentration of liposome Chinese medicine: the MPE-camptothecine is 189mM, and topotecan is that 174mM and CKD-602 are 198mM.The interior research of body of every kind of medicine will be described below.

1.MPE-the vivo medicine-feeding of camptothecine

The liposome that contains the MPE-camptothecine that long circulation is the PEG coating gives rat, and measures the blood circulation time of the medicine of liposomal encapsulated form.The liposomal encapsulated medicine and the pharmacokinetic curve of free drug are seen Figure 1A, are the function of time with the percentage ratio of injected dose.As can be seen, the blood circulation time of the type inhibitor (filled circles) of liposomal encapsulated form obviously is longer than the medicine (solid squares) of free form.For the MPE-camptothecine, the blood circulation half-life of liposomal encapsulated medicine is 14 hours, and the half-life of free drug is 50 minutes.Compare with free drug, approximately low 35 times of the rat blood clearance rate of liposomal encapsulated medicine, area under a curve is approximately high 1250 times.Analysis result shows that medicines all basically in blood flow all are retained in the liposome.

Figure 1B shows and to give behind rat Liposomal formulation (filled circles) and the free drug concentration of MPE-camptothecine in the whole blood.Long circulation time causes the drug level height in the blood.

Adopt the xenotransplantation tumor model to measure the antitumor efficient of MPE-camptothecine Liposomal formulation, wherein purebred nude mice is inoculated human colon tumor's cell HT29.Surprised is, studies show that of toxicity and antitumous effect: compare with the free drug of Isodose, it is high that MPE-camptothecine liposome toxicity is obviously wanted.Experiment and result thereof will be described below.

As the liposomal encapsulated MPE-camptothecine of embodiment 1 preparation.Carry the heteroplastic nude mice 24mg/kg of HT-29 colon, 15mg/kg and the liposomal encapsulated MPE-camptothecine of 6mg/kg dosage or the free drug of same dose.Tumor inoculation begin treatment and administration in the 10th, 16 and 23 day after 10 days.Measure during the treatment and the gross tumor volume of every animal afterwards, as described in embodiment 2.

The body weight of every laboratory animal and gross tumor volume are seen Fig. 2 A and 2B respectively, give liposomal encapsulated MPE-camptothecine 24mg/kg (filled circles), 15mg/kg (solid triangle) and 6mg/kg (solid squares) and free MPE-camptothecine 24mg/kg (open circles), 15mg/kg (hollow triangle) and 6mg/kg (square hollow) when wherein treating.

Give in the animal of liposomal encapsulated MPE-camptothecine treatment, all animals of 15mg/kg and 24mg/kg dosage group all die from the toxicity relevant with medicine after two doses, and are most of dead after 5 days in the administration first time.The animal of 6mg/kg dosage group is survival still after the 23rd day administration for the third time, ten five death of having merely hit in afterwards several days.Liposomal encapsulated MPE-camptothecine toxicity shows as the serious decline of the weight of animals, shown in Fig. 2 A.

In contrast, all animals that give the free drug treatment all survive, and have only an administration for the third time at the 23rd day in the 24mg/kg dosage group dead after several days.

Table 1

Treatment	Dosage mg/kg	The laboratory animal number	The animals survived number
						Behind the 1st dosage (the 9th day)	Behind the 2nd dosage (the 16th day)	Behind the 3rd dosage (the 23rd day)
			Normal saline	????na	????20				????20	????20	????20
Free MPE-camptothecine	????24	????10				????10	????10	????9
			Free MPE-camptothecine	????15	????10				????10	????10	????10
Free MPE-camptothecine	????6	????10				????10	????10	????10
			Liposomal encapsulated	????24	????10				????1	????0	????0
Liposomal encapsulated	????15	????10				????5	????0	????0
			Liposomal encapsulated	????6	????10				????10	????10	????5

As for the anti-tumor activity of preparation, if do not consider its big toxicity, liposomal encapsulated MPE-camptothecine and free drug specific energy mutually more effectively suppress tumor growth.By Fig. 2 B as seen, aspect the inhibition tumor growth, the liposomal encapsulated MPE-camptothecine of 6mg/kg dosage (the logarithmic growth rate is-0.026) is than free MPE-camptothecine (24mg/kg, the logarithmic growth rate the is 0.0048) significant effective of maximal dose.

The all or part of alleviation situation of monitoring experiment animal tumor the results are shown in Table 2.The alleviation fully of tumor is defined as lump disappearance when experiment finishes.The part alleviation is defined as the animal individual gross tumor volume less than 50% of maximum gross tumor volume.

Table 2

Treatment	Dosage mg/kg	Alleviate fully 1	Part is alleviated 2
				Normal saline		????0/20	????0/20
Free MPE-camptothecine	????24	????3/10	????1/10
				Free MPE-camptothecine	????15	????2/10	????0/10
Free MPE-camptothecine	????6	????0/10	????0/10
				Liposomal encapsulated	????24	????_ 3	????_ 3
Liposomal encapsulated	????15	????_ 3	????_ 3
				Liposomal encapsulated	????6	????10/10	????na 4

¹Lump disappears when all alleviating expression experiment end. ²Part is alleviated expression individual animals gross tumor volume less than 50% of maximum gross tumor volume. ³Ten animals of experimental group are all dead after the 16th day the administration second time. ⁴Na=is inapplicable

As shown in table 2, the liposomal encapsulated MPE-camptothecine of 6mg/kg dosage can make the tumor of ten animals of experimental group all alleviate effectively.Can be observed this effect in five days after the 16th day the administration second time.As noted above, the very fast death after administration for the third time of five animals in the administration group of the liposomal encapsulated medicine of 6mg/kg dosage.After experiment finishes (30 days after last treatment in the 23rd day greatly), the tumor of five surviving animals does not recur.The animal data of the MPE-camptothecine administration group that 15mg/kg and 24mg/kg are liposomal encapsulated does not provide, and is because all animals all die from the toxicity relevant with medicine, as implied above.

As shown in table 2, there is the tumor of 3 animals all to alleviate in the free MPE-camptothecine administration group of 24mg/kg, 1 part is alleviated.

Comparative drug shows with the result of free form and liposome form administration: more effective with liposomal encapsulated form administration.In fact, by relatively giving result's visible (Fig. 2 B, table 2) of the free MPE-camptothecine of liposomal encapsulated MPE-camptothecine of 6mg/kg and 24mg/kg, the effectiveness of liposomal encapsulated medicine is four times of free drug at least.The result is also clear to be shown: the dosage that the effective antitumor treatment is required, liposomal encapsulated MPE-camptothecine are wanted low four times of specific ionization medicine.

Embodiment 2 describes in detail in order to the maximum tolerated dose of determining liposomal encapsulated MPE-camptothecine and another research of minimum effective dose.In this research, press embodiment 1 described preparation liposome, give the laboratory animal Liposomal formulation by the dosage of 0.1mg/ml, 0.5mg/ml, 1mg/ml, 3mg/ml and 5mg/ml.Free drug with the 20mg/ml dosed administration in contrast.

Table 3 has been summed up every group laboratory animal number, describes the survival number each dosage different phase animal in detail.As seen from the table, all give the matched group of normal saline treatment and animal all survivals during studying of all MPE-camptothecine treatments that dissociate.Have four to die from the toxicity relevant in ten animals of the MPE-camptothecine administration group that 5mg/kg is liposomal encapsulated with medicine, one after administration for the third time the cause of the death not quite clear.A death after the administration second time is arranged, owing to lacking any corresponding toxicity sign and not owing to drug-induced in ten animals of the MPE-camptothecine administration group that 3mg/kg is liposomal encapsulated.The animal that other groups give liposomal encapsulated MPE-camptothecine treatment all survives at whole experimental session.

Table 3

Treatment	Dosage mg/kg	The laboratory animal number	The animals survived number
						Behind the 1st dosage (the 9th day)	Behind the 2nd dosage (the 16th day)	Behind the 3rd dosage (the 23rd day)
			Normal saline		????20				????20	????20	????20
Free MPE-camptothecine	????20	????10				????10	????10	????10
			Liposomal encapsulated	????5	????10				????10	????10	????5
Liposomal encapsulated	????3	????10				????10	????9	????9
			Liposomal encapsulated	????1	????10				????10	????10	????10
Liposomal encapsulated	????0.5	????10				????10	????10	????10
			Liposomal encapsulated	????0.1	????10				????10	????10	????10

Result of study is seen Fig. 3 A-3B, wherein 3A shown Mus body weight (g) with inoculation HT-29 colon tumor after the variation of natural law.Gave liposomal encapsulated type inhibitor of animal 5mg/kg (hollow triangle), 3mg/kg (hollow inverted triangle), 1mg/kg (open diamonds), 0.5mg/kg (hollow circle) and 0.1mg/kg (square hollow) and 20mg/kg (solid squares) free drug at postvaccinal the 9th, 16 and 23 day.As shown in Figure 3A, the variation of body weight is relevant with dosage, and these variations are relevant with observed toxicity.

Fig. 3 B shows gross tumor volume (mm ³) curve that changes with natural law behind the inoculated tumour, wherein consistent among dosage symbol and Fig. 3 A.Fig. 3 B shows: compare with the 20mg/kg free drug, the liposomal encapsulated MPE-camptothecine of 5mg/kg and 3mg/kg all can more effectively suppress tumor growth.It is roughly the same to give the liposomal encapsulated medicament forms (0.017 logarithmic growth rate) of 20mg/kg anti-tumor activity that free MPE-camptothecine (0.011 logarithmic growth rate) is treated and 1mg/kg.

Table 4 has been summarized the alleviation of the whole of laboratory animal and part tumor.

Table 4

Treatment	Dosage mg/kg	Alleviate fully 1	Part is alleviated 2
				Normal saline		????0/20	????0/20
Free MPE-camptothecine	????20	????0/10	????1/10
				Liposomal encapsulated MPE-camptothecine	????5	????10/10	????na 3
Liposomal encapsulated MPE-camptothecine	????3	????7/10	????1/10
				Liposomal encapsulated MPE-camptothecine	????1	????0/10	????0/10
Liposomal encapsulated MPE-camptothecine	????0.5	????0/10	????1/10
				Liposomal encapsulated MPE-camptothecine	????0.1	????0/10	????0/10

¹All alleviating expression experiment end back tumor disappears. ²Part is alleviated expression individual animals gross tumor volume less than 50% of maximum gross tumor volume. ³Na=is inapplicable

The tumor that gives the animal of the free MPE-camptothecine treatment of 20mg/kg does not reach whole alleviations.On the contrary, ten animals of the MPE-camptothecine administration group that 5mg/kg is liposomal encapsulated all show whole alleviations, and seven in ten animals of 3mg/kg dosed administration group occur all alleviating.

The result of study of embodiment 3 shows: the anti-tumor activity of liposomal encapsulated topoisomerase enzyme inhibitor MPE-camptothecine is significantly better than correlated free drug group, because the liposomal encapsulated medicine of the anti-tumor activity of 20mg/kg free drug and 1mg/kg is suitable, this shows that the antitumor effectiveness of liposomal encapsulated medicine is about 20 times of free drug.The liposomal encapsulated MPE-camptothecine of 3mg/kg and 5mg/kg is obviously more effective than 20mg/kg free drug in oncotherapy, and this shows that the therapeutic index specific ionization medicine of liposomal encapsulated medicine approximately exceeds 4-5 doubly.

2. the vivo medicine-feeding of topotecan

In supporting another research of the present invention, topotecan is encapsulated in the liposome that DSPC and mPEG-DSPC by 95: 5 mol ratios form by embodiment 4.That does not mention here studies show that in early days: topotecan is difficult for being retained in the liposome.Double-layer of lipoid is selected one pack system phospholipid for use, and the acyl chain length of this phospholipid approaches the DSPC in the mPEG-DSPC component.This bilayer has minimum packing defect, and packing defect can cause in the solid phase bilayer imperfection of adjacent contact, and the side direction of described solid phase bilayer and rotational mobility are double-deck lower than liquid.In addition, can adopt charged dextran sulfate to make topotecan be deposited on liposome interior.Other polymer, especially multi-anion copolymer such as chondroitin sulfate A, polyvinyl sulfuric acid and polyphosphoric acid also are applicable to this.

It is described to press embodiment 4, topotecan is loaded to preformed central compartment comprise in the liposome of dextran ammonium sulfate.After the load, remove by filter non-encapsulated medicine and qualitative liposome.The load liposome is 0.238 to medicine/lipoid ratio, and the liposome mean diameter is 87nm.

Vein gives liposome that rat contains topotecan to determine blood circulation time.Fig. 4 A-4B shows after the administration that the concentration of topotecan over time in the Mus blood plasma.Fig. 4 A has compared the concentration that gives to seal behind the identical 2mg/kg dosage topotecan liposome (black triangle) and free topotecan (solid squares).Fig. 4 B has compared two kinds of form medicines that give 5mg/kg dosage.The pharmacokinetic parameters of calculating sees Table 5.

Table 5

Parameter	Dosage=2mg/kg		Dosage=5mg/kg
					Free topotecan	Liposomal encapsulated	Free topotecan	Liposomal encapsulated
	?lasma?Cmax(μg/mL)	????2.89	????54.5	????8.23					????119.3
?AUC(μg/mL?h)					????0.57	????523	????1.57	????1140
	?T1/2(h)	????0.20	????7.2	????0.30					????9.8
?CL(mL/h)					????887	????0.96	????820	????1.10
	?Vol.Dist.(mL)	????173	????9.2	????278					????17.5
Elimination rate constant (l/h)					????3.45	????0.096	????2.33	????0.071

The data show of table 5, the circulation time of liposomal encapsulated medicine obviously is longer than free drug.

In another research, measured the effectiveness of liposome.Describe as embodiment 4, use liposome to the Mus of subcutaneous xenotransplantation tumor.Mice with tumor is divided into six treatment groups at random, and 12 every group, every treatment is as follows: normal saline, the MPE-camptothecine that 4mg/kg is liposomal encapsulated; The 25mg/kg topotecan of dissociating; The topotecan that 2mg/kg, 5mg/kg and 8mg/kg are liposomal encapsulated.Three dense notes administrations of vein are all carried out in all treatments once in a week, are specially the 9th, 16 and 23 day.

The tumor size of every animal of twice mensuration weekly during the research of assessment treatment effectiveness.Detect the weight of animals weekly for twice to assess the toxicity of preparation.The results are shown in Table 6 and 7 and see Fig. 5 A-5B.

Table 6

Treatment	Dosage mg/kg	Alleviate fully 1	Part is alleviated 2	No effect 3
					Normal saline		????0	????0	????12
Liposomal encapsulated MPE-camptothecine	????4	????8	????4	????0
					Free topotecan	????25	????0	????1	????11
Liposomal encapsulated topotecan	????2	????1	????2	????9
					Liposomal encapsulated topotecan	????5	????2	????8	????2
Liposomal encapsulated topotecan	????8	????7	????3	????2

¹Tumor disappears when all alleviating expression experiment end.

²Part is alleviated expression individual animals gross tumor volume less than 50% of maximum gross tumor volume.

³No effect represents that tumor is identical with initial gross tumor volume.

Shown in Fig. 5 A and table 6, untreated tumor experimental session every day with 17.8mm ³Rate increase.The tumor that gives the animal (positive controls) of liposomal encapsulated MPE-camptothecine in the experimental session rate of rise is-1.2mm ³/ day.Dosage with the 25mg/kg that is lower than 40mg/kg maximum tolerated dose (MTD) gives non-encapsulated topotecan, and the tumor propagation speed of animal is 14.1mm ³/ day.Give the animal of liposomal encapsulated topotecan treatment, wherein 2mg/kg dosage group is 0.9mm in experimental session tumor propagation speed ³/ day, the tumor propagation speed of 5mg/kg dosage group is-1.9mm ³/ day, the rate of rise of 8mg/kg dosage group is-0.8mm ³/ day.Negative growth rate representation tumor size degenerates to and is lower than initial gross tumor volume.

Calculate the size (%T/C) of the treatment tumor and the matched group tumor of all treatment groups, the results are shown in Table 6.National Cancer Institute's definition: %T/C is lower than at 42 o'clock notable antitumor activity.

Table 7

Treatment	Dosage mg/kg	％T/C 1The 29th day	%T/C the 33rd day	%T/C the 36th day
					Liposomal encapsulated MPE-camptothecine	????4	????1.8	????0.6	????1.9
Free topotecan	????25	????82.8	????79.0	????85.9
					Liposomal encapsulated topotecan	????2	????19.5	????12.9	????16.3
Liposomal encapsulated topotecan	????5	????10.5	????5.6	????5.6
					Liposomal encapsulated topotecan	????8	????2.0	????2.2	????2.2

¹%T/C is defined as the ratio of the mean tumour volume of the mean tumour volume of shown each day and the control animals that normal saline is handled.

3.CKD-602 vivo medicine-feeding

Embodiment 5 has described and has adopted that topoisomerase enzyme inhibitor CKD-602's be intended to support another research of the present invention.Employing is that the ammonium sulfate gradient of trapping agent is gone into medicine remote control medicine carrying in the liposome with the dextran.The same with the research of adopting topotecan to carry out, the molar ratio of liposome lipid composition HSPC and mPEG-DSPE is 95/5.

Fig. 6 figure shows the change curve give CKD-602 plasma concentration versus time behind the rat 1mg/kg CKD-602.The half-life that calculates liposomal encapsulated form medicine (filled circles) is that 9.8 hours and AUC are 274 μ g/mL/ hours.And the free form drug half-life that calculates be 0.2 hour, AUC is 0.37 μ g/mL/ hour.

There is the treatment of the mice assessment CKD-602 preparation of HT-20 colon cancer xenograft to render a service with lotus.72 mices that inoculated the HT-20 tumor cell are divided into six treatment groups at random.Every treated animal gives following a kind of preparation for treating: normal saline, the MPE-camptothecine that 4mg/kg is liposomal encapsulated; The free CKD-602 of 20mg/kg; The CKD-602 that 1mg/kg, 2mg/kg and 4mg/kg are liposomal encapsulated.The dense notes of vein are all carried out in all treatments once in a week three times, specifically at the 11st, 18 and 25 day.

The tumor size of every animal of twice mensuration weekly during research assessment treatment is renderd a service.Detect the weight of animals weekly for twice to assess the toxicity of preparation.The results are shown in Table 8 and 9 and Fig. 7 A-7B.

Table 8

Treatment	Dosage mg/kg	Alleviate fully 1	Part is alleviated 2	No effect 3
					Normal saline		????0/10	????0/10	????10/10
Liposomal encapsulated MPE-camptothecine	????4	????6/10	????0/10	????4/10
					Free CKD602	????20	????0/6	????0/6	????6/6
Liposomal encapsulated CKD602	????1	????2/10	????7/10	????1/10
					Liposomal encapsulated CKD602	????2	????6/10	????2/10	????2/10
Liposomal encapsulated CKD602	????4	????4/4	????0/4	????0/4

¹All alleviating expression experiment end back tumor disappears.

²Part is alleviated expression individual animals gross tumor volume less than 50% of maximum gross tumor volume.

³No effect represents that the tumor size is equal to or greater than initial gross tumor volume.

Shown in table 8 and Fig. 7 B: during studying, the tumor of normal saline treated animal is with 15.45mm ³The speed sustainable growth in/sky.The animal positive controls tumor of liposomal encapsulated MPE-camptothecine treatment is with-0.63mm ³The rate increase in/sky.Free non-encapsulated CKD 602 treated animal tumors are with 15.21mm ³The rate increase in/sky.Dosage is that the group tumor of 1mg/kg is with-2.21mm in the animal of liposomal encapsulated CKD 602 treatments ³/ day rate increase, dosage is that the group tumor of 2mg/kg is with-0.96mm ³/ day rate increase, dosage is that the group tumor of 4mg/kg is with-2.37mm ³The rate increase in/sky.Negative growth rate representation tumor size degenerates to and is lower than initial gross tumor volume.

Calculate the size (%T/C) of the treatment tumor and the matched group tumor of all treatment groups, the results are shown in Table 9.National Cancer Institute's definition: %T/C is lower than at 42 o'clock notable antitumor activity.

Table 9

Treatment	Dosage mg/kg	％T/C 1The 29th day	%T/C the 33rd day	%T/C the 36th day
					Liposomal encapsulated MPE-camptothecine	????4	????2.9	????2.3	????1.6
Free CKD602	????20	????129.1	????120.1	????99.9
					Liposomal encapsulated CKD602	????1	????11.4	????7.7	????4.4
Liposomal encapsulated CKD602	????2	????4.8	????2.8	????1.6
					Liposomal encapsulated CKD602	????4	????1.0	????1.3	????0.9

¹%T/C is defined as the ratio of the mean tumour volume of the mean tumour volume of shown each day and normal saline animals of control group.

IV. embodiment

Following embodiment is in order to preparation, characterization and the using method of the explanation present composition, but the scope that does not limit the present invention in any way.Material

Topoisomerase enzyme inhibitor (7-(4-methyl piperazine methylene)-10,11-ethylenedioxy-20 (S)-camptothecine trifluoroacetate (GI147211) (MPE-camptothecine), by the Glaxo institute, Research Triangle Park, NC provides.CKD602 (7-(2-N-isopropyl amino) ethyl)-(20S)-camptothecine) by Chong Kun Dang company, Korea S provides.Topotecan (Hycamtin ^) buy by commercially available.

The preparation material of liposome and other reagent are by commercially available acquisition.Method

Zooscopy: (Germantown's Homozygous nude mice NY) and before experiment conformed with 7 days from Taconic Farms.Animal be placed on be furnished with aseptic rodent food and acidifying water and 12:12 the daytime/night circulation isolate in the cage.Animal before inoculated tumour according to the body weight random packet.

Tumor: adopt fragment trocar implanted prosthetics (trachar placement) with growing tumors inoculation fast in the donor animal body.Cause subcutaneous xenotransplantation tumor with CCL188 HT-29.Cultured cells is through trypsin treatment, washing, counting and be suspended in 5 * 10 again ⁷In the normal culture medium of cell/ml.Tumor inoculation is by injecting 0.1ml (5 * 10 in nape portion ⁶Cell) finishes.Before begin treatment, allow tumor long to average 100mm ³Size.

Monitoring: all will observe the upgrowth situation of all animals every day.Animal will be weighed weekly before tumor inoculation and later on.The inoculation back began in 5-10 days, measured the tumor size in whole experiment weekly twice.Any compare with the beginning body weight descend 15% or above and gross tumor volume greater than 4000mm ³Animal all can not be used for research.

Embodiment 1

Seal the preparation of the liposome of topoisomerase enzyme inhibitor

The preparation of liposome and the load of selected topoisomerase are as described below.

A. liposome preparation

Lipoid hydrogenated soy phosphatidyl choline (HEPC), cholesterol (Chol) and mPEG-DSPE (ratio is 56.4: 38.3: 5.3 moles/mole) are in 65 ℃ of ethanol that are dissolved in the 250ml round-bottomed flask.In 65 ℃ of continuous stirring at least 30 minutes.The total concentration of lipoid is the total lipoid of 3.7g/10ml ethanol in the ethanol.

Dissolved class lipoprotein solution is transferred in the 250ml round-bottomed flask that contains the ammonium sulfate that 100ml concentration is 250mM (balance to 65 ℃).Continuous mixed ethanol in 65 ℃ of water-baths: lipoid: the hydrated mixture of ammonium sulfate formed few layer (oligolamellar) liposome of hydrous ethanol at least in 1 hour.

Adopt Lipex heat tube extruder to make hydrated mixture pass through the polycarbonate membrane of known pore size to reduce the particle diameter of few layer liposome.The film of mixture by 0.20 μ m aperture 5 times, the film by 0.10 μ m aperture 10 times.Moisture chamber, the inside of the liposome of extruding (one or more) sulfur acid ammonium, they are suspended in wherein the outside water content phase medium also has.Be stored in the filtration process before the remote control medicine carrying in the refrigerator through granulated liposome.

The selected topoisomerase enzyme inhibitor MPE-of 100mg camptothecine, CKD-602 or topotecan are dissolved in the concentration that forms a 2.5mg/ml in the sucrose solution of 40ml 10%.After the dissolving, solution is removed insoluble granule by the film of 0.20 μ m.

B. the remote control medicine carrying of liposome

Ammonium sulfate and ethanol before the remote control medicine carrying with the sign molecular weight be 100Kda hold back the tube by doughnut cut stream percolation from the moisture bulk phase in outside, remove.Keep constant sampling volume, and adopt seven exchange volumes liposome to be suspended in contain the outside aqueous phase of 10% sucrose at least.

After the diafiltration, liposome and medicine are so that (drug solution: the ratio of liposome) 1: 4 (v/v) mixes and heats to 65 ℃ with the moisture jacketed vessel of pre-equilibration rapidly.Mixture is lowered the temperature rapidly with ice-water bath after 40-60 minute 65 ℃ temperature maintenance.Behind the remote control medicine carrying, the crystal that exists in the liposomal samples of taking a sample to check is to determine to seal percentage mean diameter when.

Employing can be held back and be indicated molecular weight is the daltonian tube of holding back of 100K, cuts the stream percolation by doughnut and remove non-encapsulated medicine from the bulk phase medium.Adopt the exchange volume at least eight times, liposomal encapsulated medicine is suspended in contain the outside aqueous phase of 10% sucrose, 10 mM histidine pH6.5.

Final Liposomal formulation with the injection filter aseptic filtration of the acetyl cellulose of 0.22 μ m and lucifuge cold preservation until use.

C. the characterization of liposome

Seal percentage ratio and adopt the size exclusion chromatograph method to measure, promptly use recently determining of medicine in the void volume (liposomal encapsulated) and medicine total amount (void volume adds the included volume).Drug level in the pillar fraction is measured with trap.The accurate electrically laser scattering method (QELS) of mean diameter.After aseptic filtration, measure TL concentration to determine medicine/fat ratio.Preparation and characterization load have MPE-camptothecine, topotecan and 7-(2-N-isopropyl amino) ethyl)-(20S)-liposome of camptothecine (CKD-602).The results are shown in following table.

Parameter	Liposomal encapsulated MPE-camptothecine	Liposomal encapsulated topotecan	Liposomal encapsulated CKD-602
				Lot number	????221AZ43A	????221AZ43B	????221AZ53
The lipid total concentration	????17.81μmol/mL	????15.97μmol/mL	????14.079μmol/mL
				Drug level	????2.69mg/mL ????(4.55μmol/mL)	????1.72mg/mL ????(3.76μmol/mL)	????1.77mg/mL ????(3.77μmol/mL)
Medicine: fat is than (mol/mol)	????0.26(1:3.92)	????0.24(1:4.25)	????0.27(1:3.73)
				Mean diameter	????99nm	????95.4nm	????96.7nm
Seal percentage rate	????96.4％	????99.9％	????95.3％

Render a service in the body of the MPE-camptothecine that embodiment 2 is liposomal encapsulated

As the liposomal encapsulated MPE-camptothecine of preparation as described in the embodiment 1.Liposomal encapsulated medicine and free drug all are diluted to desired concn with 5% dextran aqueous solution.

As described in top method part, give nude inoculation CCL188 HT-29.70 Mus be divided at random seven groups as follows: 24mg/kg, 15mg/kg and 6mg/kg free drug group; The medicine group that 24mg/kg, 15mg/kg and 6mg/kg are liposomal encapsulated; The normal saline group.The 10th day gross tumor volume is near 75mm after inoculation ³The time begin treatment.All treat the dense notes administration of equal vein, and are weekly, and totally three times, specifically at the 10th, 16 and 23 day.

Basic evaluation standard with each experimental session and the size of tumor afterwards conduct treatment effectiveness.Measure body weight with evaluate toxicity.End the treatment back and during putting to death, observe tumor animal by above standard.When most of control tumor reach the maximum volume (4000mm that allows ³) time finish experiment.

In the tumor size of different every animals of time replication and as relevant information.Because the tumor size is relevant with the time after the administration, determination and analysis is set up each data repeatedly.Show number conversion relatively reasonable by deal with data.Represent the original measured value of tumor with Y, establish Z=log (Y+1).After the translation data, data converted Z is carried out replicate analysis.Adopt SAS program PROC MIXED.Assess every group logarithmic growth speed and the difference between more different treatment groups.Significant difference on the statistics is 0.05, but owing to be that many groups compare, need to adjust I type error and P value and show between the contrast in office that less than 0.0033 significant difference is all arranged.

The results are summarized in table 1 and 2 and Fig. 2 A-2B.

Embodiment 3

Liposomal encapsulated MPE-camptothecine Research on dose

Be encapsulated with the liposome of MPE-camptothecine as preparation as described in the embodiment 1.Liposomal encapsulated medicine and free drug all are diluted to desired concn with 5% dextran aqueous solution.

As described in top method part, give nude inoculation CCL188 HT-29.70 Mus are divided into following seven groups at random: the medicine that 0.1mg/kg, 0.5mg/kg, 1mg/kg, 3mg/kg, 5mg/kg or 20mg/kg are liposomal encapsulated; Normal saline.The 9th day gross tumor volume is near 75mm after inoculation ³The time begin treatment.All treat the dense notes administration of equal vein, and are weekly, totally three times, are specially the 10th, 16 and 23 day.

The assessment of tumor size and analyzing as described in the embodiment 2 the results are shown in Table 3 and 4 and Fig. 3 A-3B.

Embodiment 4

Render a service in the body of liposomal encapsulated topotecan

A. liposome preparation

Be prepared as follows the liposome of sealing topotecan:

Lipoid be 95: 5 distearoylphosphatidylcholine of mol ratio (DSPC) and (N-(carbonyl-methoxy poly (ethylene glycol) 2000)-1,2-distearyl-sn-glycerol-3-phosphate ethanolamine) (mPEG-DSPE), continue to stir in 70 ℃ and be dissolved in the ethanol.Lipid concn is 8.9g/10mL in the ethanol liquid.

With the dextran sodium sulfate salt is that raw material makes the dextran ammonium sulfate through ion exchange chromatography.The dextran sodium sulfate salt is soluble in water and regulate the dextran ammonium sulfate solution of pH to 5 preparation 100mg/mL with ammonia.

100mL dextran sulfate liquor is heated to 70 ℃ and merge with the ethanol liquid of lipoid, mixes forming few layer liposome.It is 70 ℃ that continuous stirring kept the temperature of the few layer liposome dispersion of hydrous ethanol in one hour.

Mixture heated to 70 ℃ after the hydration makes its mean diameter be reduced to about 100nm with a Lipex heat tube extruder by a series of polycarbonate membrane granulating.Typically, the film by aperture 0.2 μ m is 5 times successively, and the film by aperture 0.1 μ m is 10 times again.

Before the supported active medicine, from outside aqueous bulk phase, remove non-encapsulated dextran sulfate polymer and residue ethanol by carry out eight volume-exchange with the 350mM sodium chloride solution, reuse 10% sucrose solution carries out 8 volume-exchange.The diafiltration tube that adopts can be held back and indicate molecular weight is 100,000 daltonian materials.

The topotecan solution of preparation 2.5mg/mL in 10% sucrose.Drug solution and diafiltration liposome merge by 4: 1 volume ratio, and the temperature of gained mixture rose to 70 ℃ and continuous stirring one hour.Then with the load of mixture behind the rapid cooling load of ice-water bath with the termination active medicine.

To indicate molecular weight be 100,000 daltonianly to hold back a diafiltration and remove non-encapsulated medicine with holding back.Typically, with 10% sucrose 10mM histidine pH6.5 in return buffer solution carry out the exchange of 8-10 volume.

Absorb to determine drug concentrations and it is diluted to ultimate density by measuring ultraviolet-visible.Final step before the bottling comprises with the aseptic classified filtering of the filter of 0.22 μ m.

B. the characterization of liposome

Adopt the size exclusion chromatograph method to measure and seal percentage ratio, be i.e. the ratio of the medicine in the void volume (" liposome medicament ") and the total dose that from inclued and void volume, reclaims.Measure drug level with ultraviolet-visible spectrophotometer.With the electrical laser scattering method mean diameter of standard.Determine total lipoid amount with the phosphorus algoscopy.The results are shown in following table.

Parameter	Liposomal encapsulated topotecan
		TL	????17.2mg/mL
Total medicine	????2.1mg/mL
		Medicine: fat is than (mol/mol)	????0.238
Mean diameter	????87.3nm
		Seal percentage ratio	????98.8

C. pharmacokinetics and effectiveness in the body

Described in above-mentioned method part, give 72 mouse inoculation cancerous cell HT-29.Behind the tumor inoculation Ninth Heaven, a kind of intravenously administrable that animal is carried out in the following method is treated once in a week: normal saline; The MPE-camptothecine that 4mg/kg is liposomal encapsulated; The 25mg/kg topotecan of dissociating; The topotecan that 2mg/kg, 5mg/kg and 8mg/kg are liposomal encapsulated.The all weekly dense notes of vein of all treatments three times, were specially the 9th, 16 and 23 day totally.

As assessment as described in the embodiment 2 with detect the tumor size, the results are shown in Table 6 and 7 and Fig. 5 A-5B.

Embodiment 5

Render a service in the body of liposomal encapsulated CKD-602

A. the preparation of liposome and characterization

Except using the drug solution of CKD-602, other contain the liposome of CKD-602 by embodiment 4 described preparations.As mensuration liposome feature as described in the embodiment 4, the result is as follows.

Parameter	Liposomal encapsulated CKD-602
		TL	????12.5mg/mL
Total medicine	????2.07mg/mL
		Medicine: fat is than (mol/mol)	????0.315
Mean diameter	????92.8nm
		Seal percentage ratio	????94.7

B. pharmacokinetics and effectiveness in the body

Described in above-mentioned method part, give 72 Mus inoculation cancerous cell HT-29.Behind the tumor inoculation 11 days, a kind of intravenously administrable that animal is carried out in the following method is treated weekly: normal saline; The MPE-camptothecine that 4mg/kg is liposomal encapsulated; 20mg/kg CKD-602; The CKD-602 that 1mg/kg, 2mg/kg and 4mg/kg are liposomal encapsulated.The all weekly dense notes of vein of all treatments three times, were specially the 11st, 18 and 25 day totally.

Experimental session weekly the tumor size of every animal of twice detection with assessment treatment effective dose.The body weight of every animal of twice monitoring is with the toxicity of assessment preparation weekly.The results are shown in Table 8 and 9 and Fig. 7 A-7B.

Although specific embodiments of the present invention is described, those skilled in the art understand those easily and do not depart from category of the present invention at variation and modification that the present invention made.

Claims (18)

1. compositions that is used for the treatment of patient tumors, comprise the liposome that contains vesicle-forming lipids and 1-20 mole %, under polymer can be distributed in condition on the liposome duplicature both sides, form described liposome with the vesicle-forming lipids of hydrophilic polymer derivatization; With

Be encapsulated in the topoisomerase enzyme inhibitor of at least 0.10 micromole medicine/micromole's lipoid in the liposome, described liposome has the inside/outside ion gradient, is enough to make the topoisomerase enzyme inhibitor of specific concentrations to be retained in the liposome.

2. compositions as claimed in claim 1, wherein topoisomerase enzyme inhibitor is selected from the type inhibitor that comprises camptothecine and camptothecin derivative.

3. compositions as claimed in claim 2, wherein camptothecin derivative is selected from 9-aminocamptothecin, the 7-ethyl-camptothecin, 10-hydroxycamptothecine, the 9-nitrocamptothecin, 10,11-methylene-dioxy camptothecine, 9-amino-10,11-methylene-dioxy camptothecine, 9-chloro-10,11-methylene-dioxy camptothecine, irinotecan, topotecan, (7-(4-methyl piperazine methylene)-10,11-ethylenedioxy-20 (S)-camptothecine, 7-(4-methyl piperazine methylene)-10,11-methylene-dioxy-20 (S)-camptothecine and 7-(2-N-isopropyl amino) ethyl)-(20S)-camptothecine.

4. compositions as claimed in claim 1, wherein topoisomerase enzyme inhibitor is to be selected from 6-[[2-(dimethylamino)-ethyl] amino]-3-hydroxyl-7H-indeno [2,1-c] quinoline-7-ketone dihydrochloride, azotoxin and 3-methoxyl group-11H-pyridine [3 ', 4 '-4,5] pyrroles [3,2-c] quinoline-1, the type of 4-diketone/II inhibitor.

5. compositions as claimed in claim 1, wherein hydrophilic polymer is that molecular weight is 500-5,000 daltonian Polyethylene Glycol.

6. as the compositions in arbitrary aforementioned claim, wherein liposome comprises that phase transition temperature is at the vesicle-forming lipids more than 37 ℃.

7. compositions as claimed in claim 6, wherein vesicle-forming lipids is selected from hydrogenated soy phosphatidyl choline, distearoylphosphatidylcholine and sphingomyelins.

8. compositions as claimed in claim 6, wherein liposome comprises the DSPE of the poly-diethanol derivatization of usefulness of hydrogenated soy phosphatidyl choline, 1-20 mole % of 20-94 mole % and the cholesterol of 5-60 mole %.

9. compositions as claimed in claim 6, wherein liposome comprises the DSPE of the poly-diethanol derivatization of usefulness of hydrogenated soy phosphatidyl choline, 5-20 mole % of 30-65 mole % and the cholesterol of 30-50 mole %.

10. compositions as claimed in claim 6, wherein liposome comprises the DSPE of the poly-diethanol derivatization of usefulness of the distearoylphosphatidylcholine of 20-94 mole % and 1-20 mole %.

11. as each compositions among the claim 1-10, wherein comprise multi-anion copolymer in the liposome, described polymer can form complex with described topoisomerase enzyme inhibitor.

12. as the compositions of claim 12, wherein said multi-anion copolymer is selected from dextran sulfate, chondroitin sulfate A, polyvinyl sulfonic acid and polyphosphoric acid.

13. a compositions that is used for the topoisomerase enzyme inhibitor administration comprises the liposome that contains vesicle-forming lipids, and the inside/outside ion gradient of liposome is enough to make medicine to be retained in the liposome; Be encapsulated in the topoisomerase enzyme inhibitor that concentration in the liposome is at least 0.20 micromole medicine/micromole's lipoid.

14. as the compositions of claim 13, wherein topoisomerase enzyme inhibitor is the type inhibitor that is selected from MPE-camptothecine, topotecan and (7-(2-N-isopropyl amino) ethyl)-(20S)-camptothecine.

15. compositions as claim 13, wherein topoisomerase enzyme inhibitor is to be selected from 6-[[2-(dimethylamino)-ethyl] amino]-3-hydroxyl-7H-indeno [2,1-c] quinoline-7-ketone dihydrochloride and 3-methoxyl group-11H-pyridine [3 ', 4 '-4,5] pyrroles [3,2-c] quinoline-1, the type of 4-diketone/II inhibitor.

16. as the compositions of claim 13, wherein further comprise multi-anion copolymer in the liposome, described polymer energy and described topoisomerase enzyme inhibitor form complex.

17. as described above claim each compositions as the treatment tumor medicinal usage.

18. be used for the treatment of purposes in the medicine of patient tumors in preparation as each compositions of claim 1-16.

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